Protein phosphatase 2A (PP2A) is an important component of a variety of signal transduction pathways, but many of its physiological functions and substrates remain unknown. PP2A is a heterotrimer, composed of a catalytic C-subunit and a scaffolding A-subunit that mediates its interaction with a variety of regulatory B-subunits and tumor antigens. More than 15 different B-subunits have been identified, but little is known about their function or how they target PP2A to specific substrates and cellular locations. The objectives of this proposal are to investigate the roles of these different regulatory proteins in regulating PP2A function. Using a retroviral delivery system, stable cell lines have been made that can be induced to overexpress individual B-subunits or the SV40 small t antigen. When these exogenous PP2A regulatory proteins are overexpressed, the cells display an altered phenotype reminiscient of cell cycle and cytoskeletal defects. These cell lines will be used to investigate the role of the regulatory proteins in directing PP2A functions. The specific aims of the project are: 1) to determine the relative affinities of the various B-subunits and small t for the PP2A heterocomplex and their affect on PP2A localization in vivo by using immunoprecipitation and immunofluoresecence; 2) to determine how these regulatory proteins affect the architecture and phosphorylation state of the microtubule, microfilament, and intermediate filament cytoskeletal networks using immunofluorescence and immunoprecipitation; 3) to investigate the involvement of PP2A and individual regulatory subunits in select signaling pathways including MAP kinase, JNK, NF-kB, p53, and Rho using luciferase reporter assays, phospho-specific antibodies, and dominant-negative proteins.